The Tamoxifen-responsive Estrogen Receptor α Mutant D351Y Shows Reduced Tamoxifen-dependent Interaction with Corepressor Complexes*

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors ERα and β, which function as ligand-induced transcriptional factors. The nonsteroidal anti-estrogen tamoxifen is the most commonly used endocrine in the treatment of all stages of breast cancer in both pre- and postmenopausal women. Several lines of evidence have indicated that tamoxifen promotes association between ERα and corepressors N-CoR or silencing mediator for retinoid and thyroid hormone receptor (SMRT). Our results indicate that N-CoR/SMRT recognize and interact with helices H3 and H5 of the ERα ligand-binding domain in a 4-hydroxy tamoxifen-dependent manner. The mutant ERα(D351Y), derived from a tamoxifen-stimulated tumor and containing an amino acid substitution at position 351 within H3, showed reduced interaction with N-CoR/SMRT and high tamoxifen-induced activation function-1 (AF-1) activity. While the estradiol-dependent transcriptional activity of ERα(D351Y) was almost equal to that of wild-type ERα, the mutant exhibited higher levels of transcriptional activity in the presence of both E2 and 4-hydroxy tamoxifen compared with wild-type ERα. These results may explain the observation that the growth of tumor cells expressing ERα(D351Y) can be stimulated by tamoxifen, E2, or both.


The effects of estrogen and anti-estrogen are mediated through the estrogen receptors ER␣ and ␤, which function as ligand-induced transcriptional factors. The nonsteroidal anti-estrogen tamoxifen is the most commonly used endocrine in the treatment of all stages of breast cancer in both pre-and postmenopausal women. Several lines of evidence have indicated that tamoxifen promotes association between ER␣ and corepressors N-CoR or silencing mediator for retinoid and thyroid hormone receptor (SMRT). Our results indicate that N-CoR/SMRT recognize and interact with helices H3 and H5 of the ER␣ ligand-binding domain in a 4-hydroxy tamoxifendependent manner. The mutant ER␣(D351Y), derived from a tamoxifen-stimulated tumor and containing an amino acid substitution at position 351 within H3, showed reduced interaction with N-CoR/SMRT and high tamoxifen-induced activation function-1 (AF-1) activity.
While the estradiol-dependent transcriptional activity of ER␣(D351Y) was almost equal to that of wild-type ER␣, the mutant exhibited higher levels of transcriptional activity in the presence of both E2 and 4-hydroxy tamoxifen compared with wild-type ER␣. These results may explain the observation that the growth of tumor cells expressing ER␣(D351Y) can be stimulated by tamoxifen, E2, or both.
The crystal structures of several nuclear receptor ligandbinding domain (LBDs) have now been determined and reveal striking structural conservation despite modest sequence homology (39 -46). The LBD forms a structure described as a sandwich of 12 ␣-helices (H1-H12) with a central hydrophobic ligand binding pocket (47,48). In the presence of ligand, the hinge region between H11 and H12 is moved closer to H3 and H5, and H12 is positioned over the ligand binding pocket formed by H3, H4, and H5. The repositioned H12 releases the corepressors from the LBD and forms a hydrophobic groove with H3 and H5 (39,49,50). This hydrophobic groove is known to be important for interaction with LXXLL motifs found in p160 family members as well as in other coactivator molecules (51)(52)(53)(54)(55).
The estrogen dependence of some breast cancers is well known, and endocrine therapy is used to control this disease (56). The development of inhibitory ligands for nuclear receptors has yielded important therapeutic treatments, including the use of tamoxifen for the endocrine therapy of breast cancer (57). Tamoxifen exhibits a wide range of estrogen-like and anti-estrogen actions according to the target tissue examined (58). While tamoxifen may exert anti-estrogenic activity by silencing the transcriptional activity of AF-2, agonist activity of tamoxifen can be mediated through AF-1 in a cell-or tissuedependent manner (59 -63). The tamoxifen-related compound raloxifene is used clinically for the prevention of osteoporosis * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work. ‡ ‡ To whom correspondence should be addressed. Tel.: 81-3-5841-7891; Fax: 81-3-5841-8477; E-mail: junny-tky@umin.ac.jp. 1 The abbreviations used are: ER, estrogen receptor; AF, activation function; SRC, steroid receptor coactivator; TIF, transcriptional intermediate factor; TR, thyroid hormone receptor; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; HDAC, histone deacetylase; H, helix; RXR, retinoid X receptor; ID, interaction domain; LBD, ligand-binding domain; OHT, 4-hydroxy tamoxifen; RAL, raloxifene; ICI, ICI182,780; TSA, trichostatin A; GST, glutathione S-transferase; TK, thymidine kinase; ERE, estrogen response element. and is being tested for the prevention of breast cancer in highrisk women (58). However, most patients undergoing long-term treatment of breast cancer with tamoxifen eventually experience recurrence of tumor growth. One of the reasons for this treatment failure is the acquisition by the tumor of the ability to respond to tamoxifen as a stimulatory rather than inhibitory ligand (64 -67). Wolf et al. identified a mutant ER␣ from a tamoxifen-stimulated tumor that contained a point mutation that led to a tyrosine for aspartate substitution at amino acid 351 (ER␣(D351Y)), located within the LBD of ER␣ (68,69). However, the molecular mechanism of this substitution in causing tumor growth recurrence remains unclear (41, 58, 70 -76).
Recent studies have suggested that tamoxifen promotes the binding of ER␣ to N-CoR/SMRT and that the relative expression levels of coactivators and corepressors may modulate the ability of tamoxifen to regulate ER␣ transcriptional activity (77)(78)(79)(80)(81). Here, we report that N-CoR/SMRT recognized and associated with ER␣ helices H3 and H5 in the presence of 4-hydroxy tamoxifen (OHT) or raloxifene (RAL). ER␣(D351Y), derived from a tamoxifen-stimulated tumor, contains an amino acid substitution in H3 and exhibited reduced interaction with N-CoR and SMRT in the presence of OHT or RAL. In contrast, the 17␤-estradiol (E2)-dependent coactivator binding affinity of the D351Y mutant was comparable with that of wild-type ER␣. Our observations may help explain the observation that the transactivation function of ER␣(D351Y) is enhanced by both tamoxifen and estrogen.
Transfection, Luciferase Assay, Mammalian Two-hybrid Assay, and Repression Assay-For mammalian two-hybrid assays, 1 g of 17 m8luc vector was cotransfected with 250 ng GAL-DEF or mutant constructs in combination with 250 ng VP-N-CoR, SMRT, or TIF2 plasmids. For luciferase assays, 100 ng ERE-tk-luc plasmid was cotransfected with 25 ng ER␣ expression vector (HEG0) or mutants. For repression assays, 1 g of MH100-tk-luc vector was cotransfected with 250 ng GAL-DEF or mutant constructs. As a reference plasmid to normalize for transfection efficiency, 5 ng pRL-CMV vector (Promega) was cotransfected in all experiments. Six h after transfection, media were replaced with fresh medium containing 0.2% fetal bovine serum. At this time, ligands E2 (10 nM), OHT (100 nM), RAL (100 nM), ICI182,780 (100 nM), ethanolic vehicle, or 5 ng/ml trichostatin A (TSA) were added, and cells incubated for additional 12 h. Preparation of cell extracts and dual luciferase assays were performed following the man-

FIG. 1. Tamoxifen and raloxifene induce association between ER␣ and N-CoR or SMRT.
Tamoxifen-and raloxifene-induced binding between ER␣ and N-CoR/SMRT was confirmed by coimmunoprecipitation. Nuclear extracts were prepared from 293T cells coexpressing FLAG-tagged ER␣ and Myc-tagged N-CoR or SMRT. FLAG-ER␣ was then immunoprecipitated by anti-FLAG antibody, and Myc-N-CoR or Myc-SMRT in the anti-FLAG immunoprecipitates detected by immunoblotting with anti-Myc antibodies.

FIG. 2. Binding between ER␣ LBD and IDs of N-CoR or SMRT was induced by tamoxifen and raloxifene.
A, schematic representation of ER␣ and corepressors N-CoR/SMRT. B, ID1 and ID2 regions of N-CoR/SMRT were responsible for the tamoxifen-or raloxifene-dependent binding to ER␣. GST-pull down assays were performed using GST-fused ID1 and ID2 proteins produced by Escherichia coli and [ 35 S]methionine-labeled ER␣ translated in vitro in the presence of OHT (1 M). C, OHT-and RAL-induced binding between ER␣ and N-CoR/SMRT was examined using the mammalian two-hybrid system. 293T cells were transfected with 1 g of luciferase reporter plasmid bearing GAL4 binding elements (17 m8-luc) and either 0. ufacturer's protocols (Promega). Individual transfections, each consisting of triplicate wells, were repeated at least three times.
GST-Pull Down Assay-For GST-pull down assays, bacterially expressed GST fusion proteins or GST bound to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) were incubated at 30°with [ 35 S]methionine-labeled proteins expressed by in vitro translation using the TNT ® -coupled transcription-translation system (Promega). After 2 h of incubation, free proteins were removed by washing the beads with NET-N ϩ buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH7.5, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Specifically bound proteins were eluted by boiling in SDS sample buffer and analyzed by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, radiolabeled proteins were visualized using an image analyzer (BAS1500, Fuji Film, Tokyo, Japan).

RESULTS
The ER␣ Binding Surface for N-CoR/SMRT Is Located in the Helix H3 and H5-Several reports have indicated that tamoxifen promotes interaction between ER␣ and the N-CoR or SMRT corepressors (77)(78)(79)(80)(81). Therefore, we first confirmed the tamoxifen-or raloxifene-dependent interaction between ER␣ and N-CoR/SMRT by coimmunoprecipitation. Using an anti-FLAG antibody, FLAG-ER␣ fusion proteins were immunoprecipitated from nuclear extracts of 293T cells that were cotransfected with FLAG-tagged ER␣ and either Myc-tagged N-CoR or Myc-tagged SMRT. Strong anti-Myc antibody binding was observed on immunoblots of anti-FLAG immunoprecipitates from both Myc-N-CoR or Myc-SMRT cotransfectants treated with OHT or RAL (Fig. 1, lanes 3, 4, 8, and 9), whereas no coimmunoprecipitates were observed in cotransfectants treated with E2 or in the absence of ligand (Fig. 1, lanes 1, 2, 6, and 7). Weak interactions were observed in cotransfectants treated with pure anti-estrogen, ICI (Fig. 1, lanes 5 and 10).
Recently, it was shown that the RXR and TR LBDs associate with N-CoR or SMRT via the N-CoR/SMRT domains ID1 and ID2 (26,29,30,37,38) and that substitution of an isoleucine to arginine in H3 or a valine to arginine in H5 of TR or RXR, respectively, reduced N-CoR or SMRT binding (38). Therefore, to map the regions responsible for the interaction between ER␣ and N-CoR/SMRT, we first assessed whether GST-ID1 or -ID2 fusion proteins could associate with in vitro translated ER␣. A Bars indicate fold-change in luciferase activity relative to the GAL and VP vectors. Results represent the average of at least three independent experiments; error bars indicate standard deviation. C, tamoxifen-or raloxifene-dependent interactions between ER␣ mutants and N-CoR/SMRT were confirmed by coimmunoprecipitation. Nuclear extracts were prepared from 293T cells coexpressing either FLAG-tagged ER␣, ER␣(I358R), ER␣(G366T), or ER␣(D376R) and either Myc-tagged N-CoR or SMRT. FLAG-ER␣ or ER␣ mutants were immunoprecipitated by an anti-FLAG antibody, and Myc-N-CoR or Myc-SMRT in anti-FLAG immunoprecipitates detected by immunoblotting with anti-Myc antibodies.
GST-pull down assay showed that both GST-ID1 and -ID2 directly bound ER␣ in the presence of OHT (Fig. 2B) or RAL (data not shown). We then studied whether N-CoR or SMRT physically interacted with the ER␣-LBD in the presence of OHT or RAL using a mammalian two-hybrid assay. Plasmid constructs consisting of a GAL4 DNA-binding domain fused with the DEF region (i.e. the LBD) of RXR (GAL-RXR) or ER␣ (GAL-DEF) were cotransfected with either VP16 transactivation domain-fused N-CoR (VP-N-CoR), SMRT (VP-SMRT) or coactivator TIF2 (VP-TIF2) constructs into 293T cells. In the presence of OHT or RAL, a strong interaction between the DEF region and N-CoR or SMRT was observed (Fig. 2C, lanes 22, 23,  26 and 27). As reported previously, unliganded-RXR was observed to bind N-CoR/SMRT. In the absence of ligand, a weak interaction between the DEF region and N-CoR or SMRT was detected (Fig. 2C, lanes 21 and 25), whereas no interaction was observed in the presence of E2 or ICI (Fig. 2C, lanes 36, 37, 24  and 28). E2-dependent interaction between ER␣ and TIF2 was also detected by this assay (Fig. 2C, lane 38). These results indicated that the ID1 and ID2 in N-CoR/SMRT and the LBD in ER␣ were responsible for the interaction between corepressors and ER␣.
OHT Recruits Histone Deacetylases to ER␣-N-CoR and SMRT appear to be components of nuclear complexes that also include HDACs that repress transcriptional function via histone modification (33)(34)(35)(36). To investigate whether N-CoR or SMRT complexes recruited to ER␣-contained HDAC activity, we tested the OHT-or RAL-dependent transcriptional repression of GAL-DEF constructs on the basal transcriptional activity of a thymidine kinase (TK) promoter located downstream from GAL4 binding elements (17m ϫ 4). Consistent with previous studies, unliganded RXR repressed transactivation (Fig.  4, lane 25). In the presence of either OHT or RAL, basal transcriptional TK promoter activity was repressed ϳ40% when cotransfected with the GAL-DEF expression vector compared with the GAL4 control (Fig. 4, lanes 1-3). This repressional activity was inhibited by the addition of TSA, a specific HDAC inhibitor (Fig. 4, lanes 5 and 6), indicating that the ER␣-LBD recruited HDACs via N-CoR or SMRT in an OHT-or RALdependent manner. Next, the effects of mutated GAL-DEF(I358R), (G366T), and (V376R) on basal transcriptional activity were evaluated. The I358R and V376R mutations, which exhibited decreased corepressor association, also impaired the tamoxifen-dependent repression by GAL-DEF (Fig.  4, lanes 7-9, 19 -21), whereas the mutation G366T, which did not affect corepressor interaction, did not alter repression (Fig.  4, lanes 13-15). This indicated that N-CoR and SMRT com-plexes appeared to be essential for the repression of ER␣ activity in the presence of tamoxifen or raloxifene.
Amino Acid Substitution D351Y in ER␣ Reduces OHTdependent Corepressor Binding-While tamoxifen is the most commonly used endocrine treatment for all stages of breast cancer, development of resistance to the drug is common, with most patients treated with tamoxifen eventually experiencing tumor growth recurrence. Wolf et al. reported a naturally occurring amino acid substitution at position 351 in ER␣ (ER␣(D351Y)) derived from a tamoxifen-stimulated tumor (Fig.  5A) (68,69). If tamoxifen-induced corepressor binding to ER␣ was essential for the inhibition of tumor growth, it is likely that the ER␣ mutant ER␣(D351Y) would be unable to bind corepressor complexes. Moreover, the aspartate at position 351 is located within H3, which has been identified as part of the binding surface for corepressor complexes in the presence of tamoxifen (Fig. 5A). Therefore, we examined the binding of ER␣(D351Y) to N-CoR or SMRT complexes using a mammalian two-hybrid assay. In this assay, only weak ER␣(D351Y)-corepressor interactions were observed in the presence of OHT or RAL (Fig. 5B, lanes 13-18). The results of coimmunoprecipitation experiments confirmed the weak OHT-or RAL-dependent interactions between ER␣(D351Y) and the corepressors (Fig.  5C, lanes 3, 4, 8, and 9). Furthermore, GAL-DEF(D351Y) also showed reduced repressional activity against basal TK transcription (Fig. 5D, lanes 8 and 9).

Tamoxifen-induced repressional activity of ER␣ via HDAC was reduced by amino acid substitutions in H3 or H5.
Tamoxifen-induced repressional activity of GAL-DEF on basal transcriptional activity was reduced by amino acid substitutions in H3 or H5. Indicated vectors were transfected into 293T cells along with 1 g of luciferase reporter plasmid bearing GAL4 binding elements and the TK promoter (MH100-tk-luc), and 0.5 g of GAL-DEF or 0.5 g of GAL-RXRDEF in the presence or absence of OHT (100 nM), RAL (100 nM), or TSA (5 ng/ml). Bars indicate the repression rate of luciferase activity relative to luciferase activity of GAL-DEF in the absence of ligand. Results represent the average of at least three independent experiments; error bars indicate standard deviation.
Conversely, in the presence of E2, the transcriptional activities of ER␣(D351Y) and ER␣(G366T) were comparable with that of wild-type ER␣ (Fig. 6A, lanes 16, 18, and 20). However, the transactivation functions of ER␣(I358R) and ER␣(V376R) were extremely weak (Fig. 6A, lanes 17 and 19). As H3, H4, and H5 are also part of the binding surface for coactivators (39, 49 -55), including TIF2, SRC-1, and p300/CBP-interacting protein, we next tested the binding of ER␣ mutants to TIF2 in the presence of E2 (Fig. 6B). In a mammalian two-hybrid system, VP16-fused TIF2 (VP-TIF2) bound to GAL-DEF, GAL-DEF(D351Y) and GAL-DEF(G366T) in the presence of E2 (Fig. 6B, lanes 6, 8, and 10), but not to GAL-DEF(I358R) and GAL-DEF(V376R) (Fig. 6B, lanes 7 and 9), which indicated that the isoleucine at position 358 and the valine at position 376 were important for the binding of ER␣ to both coactivator and corepressor complexes and that only the D351Y mutant exhibited selective binding for coactivator complexes. These results were consistent with previous studies (58, 70 -75) and our result that the D351Y mutant was activated by both ligands (Fig. 6A, lane 25). DISCUSSION Cumulative data from several recent studies have indicated that ER␣ also binds N-CoR/SMRT in the presence of tamoxifen or raloxifene and that this interaction may be essential for the antagonistic effect of tamoxifen and raloxifene on ER␣ transcriptional activity (77)(78)(79)(80)(81). In this paper, we showed that the binding between tamoxifen or raloxifene-bound ER␣ and N-CoR/SMRT was mediated by the ID1 and ID2 domains of N-CoR/SMRT and the H3/H5 region of ER␣-LBD. We also found that amino acid residues 358 and 376 in the H3/H5 region of ER␣ were important for the binding of both coactivator and corepressor (Fig. 7) complexes. A previous paper has shown that amino acid substitutions of equivalent positions in H3 or H5 of TR and RXR also reduced corepressor binding (38). Therefore, the spectrum of corepressor interaction with tamoxifen-bound ER␣ appears to be similar to that of unliganded TR FIG. 5. Amino acid substitution D351Y in ER␣ reduces the tamoxifen-dependent binding to corepressor complexes. A, schematic representation of the amino acid substitution in ER␣(D351Y). B, the amino acid substitution D351Y in ER␣ reduced tamoxifen-induced binding to N-CoR/SMRT. A mammalian two-hybrid assay was performed using either GAL-DEF, GAL-DEF(D351Y) or GAL, with either VP-N-CoR, VP-SMRT, or VP. Indicated vectors were transfected into 293T cells along with a reporter plasmid bearing GAL4 binding elements (17 m8-luc) in the presence of either OHT (100 nM) or RAL (100 nM). Bars indicate fold-change in luciferase activity relative the GAL4 and VP vectors. Results represent the average of at least three independent experiments; error bars indicate standard deviation. C, reduction of tamoxifen-or raloxifenedependent interaction between ER␣(D351Y) and N-CoR/SMRT was confirmed by coimmunoprecipitation. Nuclear extracts were prepared from 293T cells coexpressing either FLAG-tagged ER␣ or ER␣(D351Y) and either Myc-tagged N-CoR or SMRT. FLAG-ER␣ or FLAG-ER␣(D351Y) was immunoprecipitated using an anti-FLAG antibody, and Myc-N-CoR or Myc-SMRT in anti-FLAG immunoprecipitates detected by immunoblotting with anti-Myc antibodies. D, the ER␣ amino acid substitution D351Y reduced tamoxifen-and raloxifene-dependent repressional activity. Indicated vectors were transfected into 293T cells along with a reporter plasmid bearing GAL4 binding elements and the TK promoter (MH100-tk-luc) in the presence or absence of either OHT (100 nM), RAL (100 nM), or TSA (5 ng/ml). Bars indicate the repression rate of luciferase activity relative to GAL-DEF in the absence of ligands. Results represent the average of at least three independent experiments; error bars indicate standard deviation.
and RXR, such that the binding surfaces of ER␣ for corepressors and coactivators may partly overlap.
The crystal structures of tamoxifen and raloxifene-bound ER␣ revealed that the position of H12 differed compared with that of H12 in E2-bound ER␣ and did not form a coactivator interaction surface (41,55,58). In the case of RXR and TR, H12 is not required for corepressor interaction and in fact hinders corepressor interaction (38). We also found that deletion of H12 resulted in the potentiation of OHT-bound ER␣ to bind corepressors and repress transcription (data not shown). Therefore, it is possible that the tamoxifen-or raloxifene-induced positioning of H12 with respect to H3 and H5 may be important for not only blocking coactivator binding but also forming a interaction surface with the corepressor/nuclear receptor motifs found in N-CoR and SMRT. Indeed, E2-bound LBD bind coactivators, rather than corepressors, indicating that alternate H12 configurations create different recognition surfaces for coactivator or corepressor complexes.
The Asp-351 position of ER␣ was found to be mutated to a tyrosine in the MCF-7 breast tumor cell line that showed stimulated growth rather than inhibition by tamoxifen (68,69). This D351Y mutant also exhibited increased tamoxifen-and raloxifene-induced activity of ERE-responsive genes. Since Asp-351 is located in H3, which forms part of the interaction surface for corepressors and is exposed when tamoxifen and raloxifene block AF-2, it is likely that Asp-351 participates directly in corepressor interaction. However, while the D351Y mutant exhibited reduced corepressor interaction, as expected, there may be another explanation for this phenomena. Tamoxifen and raloxifene both possess a bulky side chain extension that protrudes through the LBD surface near the base of H12 (41,55,85,86). This extension displaces H12, which rotates and folds back into the remainder of the hydrophobic cleft (41,55). Interestingly, Asp-351 can form hydrogen bonds with a tertiary amine group in the tamoxifen and raloxifene extensions, such that Asp-351 is thought to be key to the antagonistic character of these analogs (58,70,87). Thus, amino acid substitutions at Asp-351 might change the position of H12 induced by tamoxifen or raloxifene and prevent the formation of the interaction surface for corepressors.
While tamoxifen may exert its anti-estrogenic activity by silencing AF-2 transcriptional activity through the repositioning of H12 to block coactivator binding and promote corepressor recruitment, the agonist activity of tamoxifen is believed to be mediated through AF-1 in a cell-or tissue-dependent manner. Several studies have indicated that the tamoxifen-dependent AF-1 activity of D351Y mutant was increased compared with wild-type ER␣ (58, 70 -75). However, previous characterization of the D351Y mutant found that the relative affinity for estradiol or tamoxifen was unaffected. In our study we found that the additional mutants I358R and V376R exhibited higher OHT-dependent AF-1 activity. The observation that all three mutations, D351Y, I358R, and V376R reduced corepressor binding suggesting that the tamoxifen-induced AF-1 activity of wild-type ER␣ may be abrogated through the binding of corepressors (Fig. 7). In previous papers, we identified and characterized several coactivators for AF-1, DEAD-box protein p68/72 (84,88), and p300 (89). It is well known that p300 possesses histone acetyltransferase activity that modifies local chromatin structure into a transcriptionally permissive state (16). However, N-CoR/SMRT complexes contain histone deacetylase ac- tivity (33)(34)(35)(36), suggesting that AF-1 coactivator and corepressor complexes may act and/or bind competitively to tamoxifenor raloxifene-bound ER␣. According to this hypothesis, the intercellular balance between corepressor and coactivator complex levels may be critical for the agonistic or antagonistic effects of tamoxifen and raloxifene.
Our results indicated that while the mutants at positions 351, 358, and 376 all exhibited reduced interaction with corepressor complexes and enhanced tamoxifen responses to the same extent, only the ER␣(D351Y) mutant was detected in tamoxifen-stimulated tumor cell lines (68,90). This raised the possibility that while tamoxifen-dependent interaction between ER␣ and corepressor complexes may be the key event for the inhibition of ER␣ transactivation by tamoxifen, the disruption of this interaction may not be sufficient for the acquisition of a tamoxifen-stimulated growth phenotype. As ER␣(D351Y) was the only mutant showing reduced N-CoR/SMRT binding that retained comparable estrogen response and binding ability to TIF2, it is possible that both the disruption of corepressor binding and retention of the overall spectrum of ER␣/coactivator recognition and binding might be necessary for the acquisition of the tamoxifen-stimulated growth phenotype.